Method of obtaining anti-lymphoma antibodies

ABSTRACT

THIS INVENTION DESCRIBES THE METHOD OF OBTAINING ANTILYMPHOMA ANTIBODIES WHICH COMPRISES ISOLATING NUCLEOPROTEINS FROM A LYMPHOSARCOMA, INTRODUCING THE NUCLEOPROTEINS INTO A HOST, WITHDRAWING BLOOD FROM THE HOST, AND OBTAINING ANTIBODIES FROM THE BLOOD.

United States Patent 01 lice` 3,574,828 METHOD F OBTAINING ANTI-LYMPHOMA ANTIBODIES Julia McCain Lampkin-Hibbard, Oklahoma City, Okla., assignor of fractional part interest to Roderick Knott, Miami, Fla.

Continuation of application Ser. No. 261,687, Feb. 28, 1963. This application July 14, 1967, Ser. No. 653,576 Int. Cl. A61k 27/00 U.S. Cl. 424-85 3 Claims ABSTRACT 0F THE DISCLOSURE This invention describes the method of obtaining antilymphoma antibodies which comprises isolating nucleoproteins from a lymphosarcoma, introducing the nucleoproteins into a host, withdrawing blood from the host, and obtaining antibodies from the blood.

This application is a continuation of Ser. No. 261,687, filed Feb. '28, 1963, and now abandoned.

The present invention relates to cancer and more particularly to certain virus-like constituents of cancerous tissues and their ability to induce tumors and antibody formation. It has been found that certain nucleic acids extracted from tumors, which will be designated infectious nucleic acids, will induce the formation of tumors when injected in host animals, and that the tumors thus formed have dilerent characteristics from the tumors which were the source of the nucleic acids. It also has been found that nucleoproteins isolated from tumors have the capacity to induce the formation of antibodies in host animals which immunize against cancer in the host, and that anti-cancer vaccines may be made from tumors.

The tumors with which the invention is most concerned are a class of lymphocytic neoplasm which may be described as lymphosarcoma or lymphoma. Several distinguishable types of tumors have been developed within this class, but the original type from which the others were derived is designated P1798(S). This tumor was originally caused by a series of treatments of a male mouse with diethylstilbestrol-cholesterol and is fully described by Lampkin et al., Journal of the National Cancer Institute, volume 20, pages 1091-1112, June 1958.

These tumors are sensitive to certain steroids, especially 9-a-uorohydrocortisone (9-AFH), which cause regression of tumors after they are transferred subcutaneously into mice as descirbed 'by Lampkin-Hibbard in the Journal of National Cancer Institute, volume 24, pages 13524366 (1960). A second line of subcutaneouslytransferred tumors was developed from IP1798(S) which is resistant to (9-AFH) and is described by Lampkin- Hibbard, Journal of the National Cancer Institute, volurne 24, pages 1341-1352 (1960). Because of its resistance to steroids, it is designated `P1798(R). Surprisingly, this tumor is sensitive to 5-uorouracil (FU) which, though chemically unrelated to (9-AFH), causes tumor regression. (FU) does not have the same effect on the steroid-sensitive tumors P1798-(S) (Resistant to FU).

From these two types of tumor, other tumors may be derived, in accordance with one aspect of the invention.

Desoxyribonucleic acid was extracted from P1798(S) tumors in accordance with the methods described 'by K. S. Kirby in Biochemical Journal, volume 66, pages 495 (1957) and volume 70, page 260 k(1958) and by L. Kit in Archives of Biochemistry, volume 87, page 318 (1960).

Patented Apr. 13, 1971 EXAMPLE I Kirby kit method of preparation of DNA (desoxyribonucleic acid) In the case of P1798(S) infectious DN which was used to induce a mutant, JLl161 (resistant to steroid) in a new born mouse as indicated in Example II, the following steps were carried out in sequence.

(1) 38 grams of P1798(S) lymphoma (tumor) was obtained by sacrificing BALB/c mice bearing large 14 day tumors and pooling the tumors from a number of mice until there was obtained 38 to 40 grams of tumor. (The tumors were placed in a Petri dish and stored in an ice container to keep cold.)

(2) Then the tumors were homogenized in the cold with 10-16 volumes of 6% sodium p-aminosalicylate (6 grams/ cc.) for 10 minutes.

(3) An equal volume of 90% phenol (90 gm./ 100 cc.) was then added, and the mixture was stirred for 1 hour at room temperature with a magnetic stirrer.

(4) The homogenate was then centrifuged. To the aqueous phase containing the extracted nucleic acids (top layer after centrifuging) an equal volume of ethylene glycol monoethyl ether (ethyl Cellosolve) was -added and the DNA which precipitated out was dissolved in 0.01 molar NaCl.

(5) Sodium acetate was dissolved in the solution to a final concentration of 4% (4 grains/ 100 cc.) and the DNA was reprecipitated with an equal volume of ethyl Cellosolve.

(6) The reciprecipitation step was repeated and the DNA was nally dissolved in 0.01 molar NaCl.

(-7) After the addition of sodium acetate (4% final concentration) approximately 1-2 mg. of crystalline pancreatic ribonuclease (obtained from Sigma Chemical Co.) was added per gram wet weight of the original tissue and the DNA solution was incubated overnight at 4 C.

(8) The DNA was precipitated by adding an equal volume of ethyl lCellosolve and redissolved in 0.01 molar NaCl.

(9) One volume of 2.5 molar K2HPO4, one-twentieth of a volume of 33% HBPO., and 1 volume ethylene glycol monomethyl ether (methyl Cellosolve) were added and the mix-ture was shaken.

('10) Polysaccharide impurities are extracted into the bottom layer (aqueous) by this treatment.

(1l) After centrifugation, the organic phase was Withdrawn and dialyzed at 4 C. .against several changes of 0.01 molar NaCl (1.0% sodium acetate may also be used) to remove low molecular weight compounds and the enzymatically degraded RNA.

(12) After the addition of sodium acetate to a nal concentration of 4%, again 1 volume ethyl Cellosolve was added and the DNA was precipitated from solution.

(13) The DNA was washed with a solution consisting of one part of 0.15 molar NaCl and three parts of ethanol.

4(14) The DNA was dried in a desiccator containing CaCl2 in the refrigerator.

(15) The DNA that was dried in the desiccator was then weighed out in the same manner a drug would be and made up with saline so that 0.05 cc. contained a concentration of 0.125 mg. of DNA.

(16) The purity of the DNA was checked with th diphenylamine test and read in a Beckman DU spectrophotometer.

The DNA extracted as in Example I was used to induce tumors in mice as follows:

EXAMPLE II 4 extracted from the tumors which now is capable of inducing tumors after incubation with amino acids and adenosine triphosphate, as indicated below. The infectious RNA was prepared as follows:

EXAMPLE IV Mice with established P1798(R) tumors were given 4 injections of FU at a dosage of /mg./kg. at tive hour intervals. Five hours after the last injection, the mice were sacrificed to obtain the tumors.

The first six steps of Example I are repeated to isolate a mixture of DNA and RNA.

TABLE If-BIOASSAYS WITH Q-zx-FLUOROHYDROCORTISONE AND -FLUOROURACIL IN DNA DEVELOPED LYMPHOMAS 12th day 19th day Average Average tumor tumor Percent D ay Number Number diameter diameter change Comtr'ee'tment Dose of treat- Number of (cm.)5: Number (cmJi body Type lymphoma pound initiated mgJkgm. ments mice tumors S.E. tumors .E. Weight JL1161 (DNA) gen. 21 Control 1 7 7 1. 85:0. 17 4 3. li. 09 +12. 4 FU 1 15 10 20 0 0 0 +1. 3 FU 12 25 5 9 9 2 15:0. 08 0 0 -15. 4 Q-AFH 12 25 5 9 9 2 05:0. 10 6 2. 95:0. 05 +8. 5

EXAMPLE III The nucleic acid RNA is then purified Iby incubating Transplantation of JL-l161 lymphoma All transplanting of tumors is carried out with complete sterile technique. The Petri dishes, saline (physiological) and instruments are autoclaved for minutes to prevent any bacterial infection of the tumor. Fourteen day old tumors are excised from BALB/c mice after sacrificing and placed in a sterile Petri dish. The tumor tissue is then sliced -with a scalpel (sterile) to 1.5 mm.3 size and placed with sterile forceps into a 13 gauge trocar needle. The tumor inoculum is then placed subcutaneously on the right side of a BALB/c mouse (inbred) with the 13 gauge trocar needle. After the tumor inoculums are cut to the proper size, saline is poured on top of them to prevent drying. All tumors are transplanted before 30 minutes time after the sacrifice of the mouse to prevent drying and insure 100% takes of tumor.

This tumor, unlike the one from which it was derived (P1798(S)) was found resistant to steroids but sensitive to iiuorouracil. Mice in which .TL-1161 had been transplanted developed tumors and the mice were then treated 'with daily doses of 25 mg./kg. of (9-AFH) or (FU) with results as indicated in Table 1.

Ribonucleic acids extracted from P1798 tumors behave dilerently and do not induce tumor formation. However, it has been found possible to render RNA from P1798( R) tumors infectious The host animals whose tumors have RNA are treated with FU. Shortly thereafter, before the tumors regress, the animals are sacrificed and RNA is with 2 mg. of deoxyribonuclease per gram wet Weight of tumor tissue (Sigma Chemical Co.) for twelve hours to remove any of the DNA, at 4 C. Until this point both DNA and RNA are present. The important step is the enzyme incubation. When ribonuclease is used, this destroys RNA and leaves pure DNA and, when deoxyribonuclease is used, this destroys DNA and leaves pure RNA.

Ten mg. of the RNA preparation was incubated for days with 10 mg. arginine, 10 mg. of D and L tryptophan and 10 mg. of adenosine triphosphate in 50 cc. of medium 199 to activate the RNA to become infectious. This long incubation at 37 C. demonstrates absolutely that no cells Iwere present and that an agent was present which acted as a virus.

BALB/c mice were given 5 injections of 0.1 mg. of the resultant activated RNA preparation (in 0.5 cc. of medium 199) intraperitoneally at intervals of three days and tumors designated JL762 and JL862 were observed which are compared in Table 2. In general, rthe tumors were sensitive to 9-AFH and resistant to FU, the opposite of the sensitivity of the tumors from which they were derived. A male mouse in one of the test groups containing 8 adult mice developed lymphocytic ascites which was accompanied by extensive metastasis of lymphoma cells in the mesentary. There was no evidence of a large liver or spleen. As shown in Table 2,

TABLE 2.-BIOASSAYS WITH x- FLUOROHYDROCORTISONE AND -FLUOROURACIL IN RNA DEVELOPED LYMPHOMAS Day Average Treattumor Percent ment Number diameter Average tumor Change initi- Dose, of treat- Number Number (cm.) Number diameter (cm.) body Type lymphoma Compound ated mg./kgm. ments mlce of tumors iS.E. of tumors S.E. weight 8th day 13th day JL762 (RNA)x Control 8 8 8 1. 655:0. 05 7 2. 4i0. 11 +28. 3 Q-AFH 8 25 5 9 9 1. 75:0. 04 2 0. 16 (7 mice neg.) 14. 7 FU 8 25 5 9 9 1. 65:0. 02 9 1. 65:0. 04 12. 9

10th day 15th day JL762A (RNA)I Control 10 8 8 1 710. 05 8 2. 55:0. 07 +30. 9 Q-AFH 10 25 5 19 19 1. 65:0. 03 5 0. 4i0. 28(14 neg.) -12. 7 6,9a-difluoro 10 15 5 9 9 1. 5+ 0. 04 0 0 16. 6

16a-methyl prednisolone; U 10 25 5 7 7 l. 6&0. 11 4 1.9i0.04 (3 neg.) -13 8th day 13th day JL862A (RNA)3 Control 8 4 4 1.5=*=0. 07 4 2.7*0.09 21.0 JvAFl-I 8 25 5 5 1.610. 09 2 0.4 (31mg.) -26 F 8 25 5 4 4 1.5*007 4 1.810.05.- -2

See footnotes at end of table.

TABLE 2.-BIOASSAYS WITH Q-a-FLUOROHYDROCORTISONE AND -FLUO ROURACIL IN RNA DEVELOPED LYMPHOMAS Day Average Treattumor Percent ment Number diameter Average tumor Change initi- Dose, of treat- Number Number (cm. Number diameter (cm.) body Type lymphoma Compound ated mg./kgm. ments mice of tumors $5.13. of tumors S.E. weight 8th day 13th day .TL862B(RNA)4 Control 8 4 4 1. 15:0. 07 9-AFH 8 25 5 4 4 1. 25:0. 07 FU 8 25 5 4 4 1. 4i0. 10

14th da;r 19th day JL762(gen. 14)5 9 1. 85:0. 02 8 2. 35:0. 05 Control 14 9 [-20 ruezmgen. 14) 9 1810.13 s 2. sin. oe JL762 (gen. 14) 10 1. 95:0. 07 7 0. 2 (3 Q-AFH 14 25 5 10 -32 JL72A (gen. 14) 10 1. 85:0. 06 2 0. 2 .'IL762 (gen. 14) 9 1. 95:0. 05 9 1. 7i FU 5 9 -25 IL762A (gen. 9 1. 95:0. 07 9 1. 75:0. 04 .TL862A (gen. 12 9 9 2. 15:0. 04 7 6 2. 75:0. 08.... +23 J L862 (gen. 9 2. 05:0. 05 6 2. 25:0. 07 JL862 (gen. 2) 10 10 2. 110.05 4 0.2 (5 neg.) `43 JLSZB (gen. 2. 05:0. 08 3 0. 2 (6 neg.) JL862A. (gen. 9 9 2. 25:0. 06 5 5 2. 05:0. 10 12 J'L862B (gen. 9 2. 05:0. 07 5 l. 95:0. 08

l Tumor developed 39 days after first iniection of RNA, (male). Tumor developed 46 days after rst injection o RNA, (female). 3 Tumor developed 63 days after first injection of RNA, (male). 4 Tumor developed 63 days after rst injection of RNA, (female).

J L762 lymphoma was inoculated on the right and JL762A lymphoma was inoculated on the left side of the same mouse. 6 JL862A lymphoma was inoculated on the right and JL862B lymphoma was inoculated on the left side of the same mouse. 7 Three mice died with large tumors on both sides before the 19th day.

8 Four mice died with large tumors on both sides before the 19th day.

this activated RNA-developed tumor, a JL762 lymphoma, was bioassayed with 9-AFH and FU and found to be completely sensitive to 9-AFH and resistant to FU. Since the original RNA was extracted from P1798 (R) tumor (sensitive to FU) these data indicate that the pretreatment of RNA with FU altered the RNA and produced a 9-AFH sensitive tumor which was similar to the original P1798 (S) tumor. Another mouse from the same group developed a lymphoma 24 days later and was bioassayed with 9-AFH and FU. This lymphoma is referred to in Table 2 as JL862A. IL862A lymphoma was also found to be sensitive to 9AFH. Another RNA tumor, JL762A, developed in a female mouse in a group of 5 adult mice and was bioassayed in the same manner. TWO kinds of lymphoma cells were observed upon making a smear of ascites. Both cells are characteristic of a lymphocytic neoplasm. The smaller cells are probably sensitive to steroids since JL762A is primarily a sensitive to 9- AFH tumor and consists mostly of a large nucleus and some pseudopodia, with a small amount of cytoplasm. The two larger lymphocytic cells are probably the ones resistant to steroid (sensitive to FU). This was indicated since some tumors sensitive to FU grew upon a first generation transfer of JL762A lymphoma. The nuclei are larger and more cytoplasm is present. The 9-AFH given at a dose of 25 mg. per kg. caused essentially complete regression of established tumors in 19 mice. Furthermore, 60:,9a-difluoro-la-methyl-prednisolone, given at a dose of mg. per kg. also caused complete regression of JL762A lymphoma, thus indicating again that this RNA tumor was sensitive to steroid. However, in this case, 4 out of 7 mice showed resistance when FU was given at a dose of mg. per kg. and 3 mice were completely sensitive (mice living 130+ days) to FU. These data indicate that possibly some of the (R) tumor cells (sensitive to FU) were present in the original population of cells of JL762A. Another female mouse from the same group developed a lymphocytic ascites and was labeled ]L862B. Metastasis in the mesentary and right kidney was found. Bioassay with 9-AFH and FU showed IL862B -to be sensitive to 9- AFH. Another male mouse died of lymphocytic ascites in 71 days (3 out of 8 mice).

The IL762 and JL762A lymphomas were bioassayed with 9-AFH after being passed in series in transplant without any drug treatment. The 9-AFH was given at a dosage of 25 mg. per kg. to mice bearing both JL762 and JL762A in the same host on the 14th day after inoculation of these neoplasms. After five days of treatment with 9-AFH, the IL762 and JL762A which had changed from a resistant to a sensitive tumor regressed almost completely and has remained stable and irreversible after 14 transfer generations. This would indicate a permanent mutation has occurred. IL862A and JL862B lymphomas were inoculated into the same host and treated in the same manner with 9-AFH. Likewise a mutation from a resistant to a sensitive tumor occurred in neoplasms JL862A and JL862B. This change has remained stable and irreversible after 12 and 10 transfer generations respectively. (See Table 2.)

As noted previously, it has been found that, in addition to the nucleic acids derived from tumors being capable of inducing cancer in a virus-like manner, nucleoproteins from a variety of the types of tumors can induce antibody formation.

Nucleoproteins are isolated as follows: Tumors of the P1798 (Si) or (R) types are isolated untreated or pretreated With drugs. When a pretreatment is used FU is injected subcutaneously at a dose of 25 mg. per kg. every 6 hours, to BALB/c mice bearing large established (R) tumors (sensitive to FU) for 24 and 48 hours intervals, and then the mice Were sacrificed to obtain the tumors. 9-AFH was injected subcutaneously at the same dosage and manner to BALB/c mice bearing large established (S) tumors and then sacrificed to obtain the tumors. The tumors are weighed and homogenated in a Waring Blendor with ice cold distilled water to keep from denaturing proteins. The homogenate is centrifuged for seven minutes at 3000 r.p.m, and the supernatant decanted and further fractionated by passing through a xSeitZ lter. The precipitate containing the nucleoproteins from the tumor is further washed three times with cold distilled water. The hypotonic properties of the water splits the cell. After washing, the precipitate of the nucleoproteins is resuspended in physiological saline at a concentration of 10%. Similar procedures may be used with all of the above types of tumor and have also been used with human tumors including adenocarcinoma from the colon and breast carcinoma freshly obtained by operation.

The saline solutions of nucleoproteins were injected intrapertitoneally into rabbits biweekly (21/2-4 weeks) for 7 the production of antibodies and antibody neutralization tests have confirmed their effect. Antisera can be obtained in the same way from C57BL mice and Fischer rats. Antibody neutralization tests were used to confirm the presence of antibodies, similar to the tests described by 8 treated with FU as described previously. The control groups of mice challenged with P1798(S) or (R) had progressively growing neoplasms on the 14th day. The same Itype of 100% inhibition of tumor formation of P1798(S) or (R) or JL1161 was observed. Some of this Friend in the Journal of Experimental Medicine, volume antisera was used to test for inhibition of IRC/741 Dun- 109, page 217 (1959) except that solid P1798 and other ning leukemia in Fischer rats. However, in this experilymphomas were used instead of the leukemie spleens ment no tumor inhibition was observed and the averemployed by Friend. age tumor diameter in 8 rats was 2.4104 cm. which was The antisera from the rabbits are obtained by drawcomparable to controls. 100% of these rats treated with ing the blood directly from the rabbits heart (to obtain antisera died with Dunning leukemia. Results are shown pure non-contaminatcd blood). The blood was allowed in Table 4. to clot in Iice; and then centrifuged. The clear supernatant Formalinized vaccines were prepared by combining a serum was then placed in an incubator at 56 C. for 30 11500 dilution of formaldehyde with a 20% solution of minutes to inactivate any substances that might destroy 15 tumor homogenate of P1798(R) tumor pretreated with the activity of the antibodies. (FU). J'L1161 lymphoma vaccine was prepared in the The test used in the neutralization of antibodies consame manner but was not treated with (FU). These vacsists in making a tumor homogenate in saline sepcines lwere injected biweekly intraperitoneally at a dose of arately from three different lymphoma lines: P1798(S), 0.5 cc. The mice were challenged with tumor, as indicated P1798(R) and JL1161 lymphomas. An equal volume of 20 in Table 5, the 11th day after the last injection of the vac- 20% tumor homogenate is added to an equal volume of cine. The challenge consists of inoculating the BALB/c undiluted serum and incubated for 11/2 hours at 36 C. mice with 1.5 mm.3 of tumor with a 13 gauge trocar. The Two controls are run simultaneously: one by incubating JLl 161 lymphoma was inhibited 3.0 times more with vac- 20% tumor homogenate without antiserum (this tumor cine prepared from the JLll61 than they were with the homogenate causes 100% tumors when injected into 25 P1798(R) lymphoma. However, some cross-sensitization BALB/ c mice), the second using untreated rabbit antiwas demonstrated against the IL1161 tumor when formalserum (100% takes of tumors was obtained with (S) inized vaccine was prepared from P1798(R) tumor (preand (R) tumors). Results are shown in Table 3. treated with FU as described previously) and compared Comparable results were obtained in a study using to the P1798(R) tumor receiving the same vaccine. The Fischer rats to produce antisera. These antisera were tumors in the mice challenged with IL1161 and treated prepared and obtained by pooling sera from rats after 8 with vaccine were inhibited 3.8 times more than the P1798 injections of nucleoproteins from P1798(R) tumor pre- (R) series.

TABLE 3 [The use oi antisera obtained from rabbits injected with antigens from lymphomas, adenocarcinomas and one breast carcinoma to inhibit the growth of lymphocytic neoplasms of P1789 sensitive, P1798 resistant, and J L1161.]

Eiect of antisera on tumor formation 14th day Number treat- Day of Average ments of challenge tumor I v t l antigen Number Number after last diameter Tumors used Antisera prepared from the antigenic types l given of o mice injection Number (cm in challenge shown, (intraperperitoneally in rabbits) biweekly rabbits in group of antigen of tumors i S.E.

P1798 S 1 Control Untreated 0 1 7 (b) 7 2 1in, 03 d0 0 1 10 (b) 10 2. 5=1=0. 07 JL1161 -do 0 1 10 (b) 10 2. 3x0, 06 P1798 S or R 3 Untreated tumor4 homogenate, P1798 R 5 1 4, 8 18 0 0 P1798 S or R FU Pretrcated tumor t homogenate, (P1798 R) 6 1 8,10 16 0 0 P1798 S, R or JL1161. FU pretreated nucleoproteins, (P1798 R 9 1 8 13 0 0 P1798 S or R Untreated tumor' homogenate, (P1798 S) 02 1 5 14 0 0 P1798 S or R 9-AFH pretreated tumor homogenate, (P1798 S). 3 1 8 16 0 0 P1798 S, R or JL1161. Q-AFH pretreated nucleoproteins, (P1798 S 9 1 8 13 0 0 P1798 S, R or JL1161... FU nl 9AFH pretreated nucleoproteins (1798 R 9 1 8 13 0 0 an J L1161 Untreated tumor homogenate of JL1161 6 2 1 6 14 0 0 P1798 S, R or JL1161... Nulproteins from house fly larvae, (fed on 5 1 5, 5, 12 14 0 0 y P1798 S or R Nucleoproteins from human 7 adenocarcinoma of 8 1 5 15 0 0 colon, (JL-H-G2) J L1161 Nucleoproteins from human adenocarcinoma of 7 1 5 14 0 0 colon, (J L-H-62) P1798Sor RJL1161 Nucleopi'oteins from humanB adenocarcinoma of 7 1 5 14 0 0 colon, (JL-H-2A) P1798 S or RJ L1161.. Nucleoproteins from humang breast carcinoma, 5 1 5 14 0 0 1 P1798 lymphoma sensitive to steroid (S). 2 P1798 lymphoma resistant to steroid (R) (sensitive to FU). 3 Animals were inoculated with either P1798 sensitive or resistant lymphomas or as shown in the table.

4 Experiment was repeated with two injections to a rabbit (one 12 days after the other). Mice (5 per group) were challenged with P1798 sensitive and resistant tumors 14 days after the last injection of antigen. 100% inhibition of tumors was obtained.

Experiment was repeated with 17 mice per group and antisera completely inhibited the P1798 (R) tumors.

6 Two injections (one 12 days after the other) were given and the mice were challenged 14 days alter the last injection.

'l Experiment was repeated on two rabbits using 7 injections of nucleoproteins. Two groups of mice (8 and 5 respectively) were challenged with P1798 (R) and one group (5 mice) was challenged with P1798 (S). Antisera completely inhibited these tumors.

8 Experiment was repeated with 5 mice per group and antisera completely inhibited the tumors. Experiment was repeated and identical results were obtained. Two additional groups oi mice (8 and 13 respectively) were challenged with P1798 (R) and antisera and no tumors developed.

N o'rE.--- The subcutaneous cliallengesloi tumor were accomplished by inoculation with 0.2

gauge needle in the right flank. Control mice were given 0.2 ml. in the same manner except serum sterile syringe and 22 ml. oi a 20% tumor homogenate and antisera (1:1) with a was obtained from untreated rabbits; b Control mice challenged with tumor ar the same time as the experimental groups.

TABLE 4 The use of antisera pooled from rats injected with antigens from P1798 resistant tumor pretreated with -uorouracil to inhibit the growth oi lymphocytic neoplasms of P1798 sensitive, P1798 resistant and JL1161] Eect of antisera on tumor formation e Number 14th day Number of rats treatrecelving Day of Average ments of antigen, Number challenge tumor antigen treated of mice after last diameter Tumors used Antisera prepared from the antigenic types given or unor rats injection Number (Cm.) in Challenge shown, intraperitoneally in rats weekly treated in group o antigen of tumors =|=S.E.

P1798 S 1 Control Untreated 0 10 3 8, mice (b) 8 2. 3=*=0. 03 P1798 R 2 do 0 l0 3 5, mice (b) 5 2.720.05 P1798 S, R or .T Llll Nucleoproteins 4 from FU pretreated P1798 R.. 8 10 5 8, mice 16 0 0 IRC/741 Dunuing Nucleoproteins 4 trom FU pretreated P1798 11....- 8 10 5 8, rats 16 8 2. 4=0. 04

leukemia in rats. IRC/741 Dunning Control Untreated 0 0 4, rats 0 4 2. 3520. 13

leukemia in rats.

l P1798 lymphoma sensitive to steroid (S). 2 P1708 lymphoma resistant to steroid (R).

3 Ten normal rats were used as controls. The serum from these rats was pooled and one-half of antiserum was used for the P1798 (S) and one-half for the P1798 (R) challenge.

4 Nucleoproteins from P1798 resistant tumor pretreated with FU given to Fischer rats. 6 Antisera from 10 rots pooled and one-half used for rats and one-half used for mice.

Normal rats were used as control.

NOTE .-e The subcutaneous challenges of tumor were accomplished by inoculation with 0.2 ml. of a tumor` homogenate and antisera (1: 1) with a sterile syringe and 22 gauge needle in the rlght ilank. Control mice were given 0.2 ml. in the same manner except serum was obtained from untreated rats, b Control mice were challenged with tumor at the same time as the experimental groups, see methods.

The following experiment relevant to the above vaccine was performed in which nucleoproteins isolated from P1798(R) were given directly to BALB/c mice as a vaccine and inhibition of JL1161 lymphoma -was obtained (known as cross-sensitization) after the mice were challenged with JLL1l61 but not when challenged with -P 1798 (R) tumor.

Cross-sensitization was determined by challenging 4 ferent) protein. The cross-sensitization studies indicate that the nucleoproteins isolated from the JLlll lymphoma are similar to the abnormal protein formed when PU is given to Pl798(R) tumor. It will be noted also that the JL1161 lymphoma had more RNA per gram Wet weight of tissue than the P1798(R) tumor. This may indi- 0 cate the RNA is structurally different and acts as a template to influence the formation of a different sequence of TABLE 5 [Efect of foimalinized Vaccines obtained from P1798 lymphoma and .'IL1161 lymphoma on the growth of these two type lymphomas] Tumors after 10 days Day ot Average Number challenge tumor Tumor injections, Number after last Number diameter Vaccine challenged biweekly mice injection tumors cm.iS.E.

JL162, from JLlll JL1161 6 8 11 3 0. 7;|;0. 05

.TL162, from JLlll P1798 resistant- 6 4 11 4 2.1:i:0. 13

.TL101, rtm FU treated .TLlll 11 10 11 10 0. 5:1;0. 06

.T L101 grtm FU treated P1798 resistant 11 8 11 8 1. 910. 11

No vaccine, control .TL1161 8 (1) 8 1. 75:0. 07

No vaccine, control P1798 resistent l0 (l) 10 1. 8:l:0. 06

i Control mice of the same age of those treated with vaccines were challenged with tumor at sth eame time as the experimental groups.

groups of mice of -8 each with two tumors: JLll61 lymphoma (one group the untreated controls and the other receiving the P1798(R) nucleoproteins pretreated with FU) and the P1798(R) lymphoma (one group the untreated controls and the other receiving the P1798(|R) nucleoproteins pretreated with FU). lIncreased cross-sensitization was shown when BALB/c mice were given 6 injections at one 'week intervals using a vaccine of P1798 (R) nucleoproteins pretreated with FU and challenged with J=Lll6l lymphoma on the 9th day after the last injection. Seven out of eight mice as shown in IFIG. 1, Were completely negative to the 11.1161 protein lymphoma challenged group, but one mouse had an average tumor diameter of v0.1 cm. when challenged with a 1.5 mm.3 inoculum. [In contrast, the P1798(R) lymphoma challenged group treated in the same manner as the J-Lll61 group possessed large established tumors on the 11th day. The average diameter of these tumors on the 11th day was 1.3 cm. The JLl16l untreated control group and the P1798(R) untreated group had progressively growing neoplasms on the 11th day (1.7 cm. and 1.6 cm. average tumor diameter respectively). The inhibition of the tumor formation in mice challenged with JL1161 over that observed Iwith the P1798(R) tumor indicates that these tumors are immunologically different. In mice treated in such a manner, increased cross-sensitization was shown with the JLll61 lymphoma which lwas inhibited in its growth further indicating the formation of abnormal (difamino acids and protein composition in the JL1161 than in the P1798(R) lymphomas.

The invention represents a substantial advance on the road to ultimately eliminating cancer. The best treatment of cancer is its prevention. In the above description, it has been shown in animals that nucleoproteins isolated from human an animal tumors cause antibodies to be formed when injected into a host, which antibodies are elfective against mouse lymphomas. The neutralization test of antisera obtained from rabbits inoculated with nucleoproteins demonstrates 'that antibodies were formed. It should be possible, further, to purify these nucleoproteins by splitting them into acid soluble proteins histones and insoluble proteins, so that some day a type of protein can be given as a vaccine to prevent cancer. The fact that nucleoproteins given directly to mice as a vaccine inhibited lymphoma used as an active inoculum indicates that subcellular parts of cells may be useful to produce antibodies.

Another application would be that an animal with a tumor could have a biopsy, and that nucleoproteins could be isolated from the cancerous tissue thereby obtained which would be given to rabbits. Antisera would form in the rabbits and be given back to the animal which had the biopsy since the antibodies formed would be specific for him.

In addition, the techniques described for inducing the formation of tumors with nucleic acids previously al- 11 tered with drug treatment is useful in making mutant strains from which different nucleoproteins could be isolated and given as a vaccine.

It will be appreciated that the above-described invention has still further applications in the use of immunological techniques in the prevention and treatment of cancer and that various changes and modications may be made in the methods described without departing from the scope of this invention, as this is set forth in the claims.

What is claimed is:

1. The method of obtaining antibodies which are capable of inhibiting the formation of lymphoma in mice comprising (1) isolating nucleoproteins from a lymphosarcoma by (a) homogenizing said lymphosarcoma with ice cold distilled water, (b) centrifuging the hymogenate, (c) decanting the aqueous supernatant from the centrifuged hymogenate, (d) precipitating the nucleoproteins, (e) removing the supernatant liquid from precipitated nucleoproteins, (f) washing the precipitated nucleoproteins with cold distilled water, and (g) suspending said precipitated nucleoprotein in a saline solution, (2) intro` 12 withdrawing blood from said host and (4) separating blood serum containing said antibodies from said blood.

2. The method as claimed in claim 1 wherein the nucleoproteins are obtained from a breast carcinoma.

3. The method as claimed in claim 1 wherein the nucleoproteins are obtained from adenocarcinoma.

References Cited Batchelor et al.: The Lancet, No. 7333, p. 613, Mar. 14, 1964.

Hilleman: Wenner-Gren Center International Symposium Series, vol. 6, pp. 113 and 114, 1966.

Kidd: J. Experimental Medicine, vol. 83, pp. 227 and 228, 1946.

McAllister et al.: I. National Cancer Institute, vol. 21, pp. 549 and 550,1958.

Mohos et al.: Experimental Medicine, vol. 105, pp. 233 and 234, 1957.

Seegal et al.: Federation Proceedings, 1957, page 88.

RICHARD L. HUFF, Primary Examiner U.S. Cl. X.R. 424-88, 

